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研究生: 葉上瑜
Yeh, Shang-Yu
論文名稱: 探討與胃黏膜腸上皮化生變異相關的微小核糖核酸作為胃癌預測的生物標記
To explore intestinal metaplasia related microRNAs as biomarkers for gastric cancer prediction
指導教授: 曾大千
Tseng, Ta-Chien
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 生物科技與產業科學系
Department of Biotechnology and Bioindustry Sciences
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 74
中文關鍵詞: 胃癌 、腸上皮化生 、微型核糖核酸 、生物標誌物
外文關鍵詞: gastric cancer, intestinal metaplasia, miRNA, biomarker
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  • 根據WHO的統計,胃癌在全球的癌症發生率及死亡率均在五名內,由此可知胃癌為一個不容小覷的癌症。胃癌有95%以上是腺癌,依照Lauren’s組織學分類可分為腸道型、瀰漫型及混合型,其中以腸道型的比例佔一半以上,胃黏膜腸上皮化生(intestinal metaplasia; IM)為腸道型的癌前病變,導致其病變的因素很多,尚無完全有效的治療方法,所以胃癌的早期檢測預防及治療方法至今仍為一大挑戰。微型核糖核酸(miRNA),是一種單股且非編碼蛋白質的小片段RNA,近年來發現miRNA與繁多疾病息息相關。因此,本研究收集了患有IM的患者的組織和已康復患者的組織,透過高通量定序比較兩組間miRNA及基因表現的差異。本研究發現miR-200b-3p,miR-200c-3p,miR-10b-5p和miR-425-5p,在胃黏膜腸上皮化生組織中有異常表達的現象。利用PMA刺激胃癌細胞發炎來驗證miRNA在胃癌細胞中的表現,確定miR-425-5p的表現會增加,並確認其目標基因為BANP和RABAC1,這兩個基因在腸上皮化生組織中也有下調現象,此外抑制miR-425-5p的表達後,胃癌細胞生長及遷移會趨緩,並推測miR-425-5p可能會促進為癌發展。期望未來藉由檢測miR-425-5p的表達,嘗試開發成為一種早期診斷胃癌的非侵入式生物標記檢測平台。

    Gastric cancer is the top five incidence and mortality rates of cancers in the worldwide. More than 95% of gastric cancer is adenocarcinoma, which is divided into intestinal, diffuse and mixed histotype according to Lauren's histological classification. Intestinal type is the major of gastric adenocarcinoma, which developed from the intestinal metaplasia (IM) of precancerous lesions. Many factors cause intestinal metaplasia and there is no completely effective method to reverse it. Therefore, the early detection and the prevention of gastric cancer is still a big challenge. In recent years, miRNAs are reported to be involved in many cancer progression. Therefore, this research collected the tissues from the patients who continually suffered from IM and IM-regression after H. pylori eradication. The differential expressed miRNAs and mRNAs were identified. It has found that miR-200b-3p, miR-200c-3p, miR-10b-5p and miR-425-5p are up-regulated in the intestinal metaplasia tissue, and discovered that they were also up-regulated under inflammatory stimulation in gastric cancer cells. It was determined that the target genes of miR-425-5p were BANP and RABAC1, which were also down-regulated in intestinal metaplasia. Besides, knockdown miR-425-5p attenuated proliferation and migration of gastric cancer cells. It suggested that miR-425-5p may promote the development of gastric cancer. In the future, this research can further verify the role of miR-425-5p in intestinal metaplasia by more clinical samples, and try to develop a non-invasive biomarker detection platform for early diagnosis for gastric cancer.

    中文摘要 I 英文摘要 II 誌謝 VI 目錄 VII 表目錄 X 圖目錄 XI 附圖目錄 XII 縮寫表 XIII 一、研究背景 1 1-1 胃癌 1 1-2 腸上皮化生 2 1-3 腸上皮化生的分子特徵 4 1-4 微小核糖核酸(MicroRNA; miRNA) 5 1-5 微小核糖核酸(miRNA)與腸上皮化生 6 1-6 Phorbol myristate acetate (PMA)的應用 7 1-7 BTG3 associated nuclear protein (BANP)簡介 7 1-8 Rab acceptor 1 (RABAC1)簡介 8 1-9 選擇miRNA作為生物標記的原因 9 1-10研究目的 10 二、材料與方法 12 2-1 細胞培養 12 2-2 小分子干擾核糖核酸轉染 12 2-3 核糖核酸萃取 13 2-4 反轉錄聚合酶連鎖反應 14 2-5 定量即時聚合酶鏈鎖反應 14 2-6 微小核糖核酸反轉錄聚合酶連鎖反應 15 2-7 微小核糖核酸定量及時聚合酶反應 15 2-8 質體建構 16 2-9 質體轉染 17 2-10螢光素酶(luciferase)報導基因分析 18 2-11細胞生長試驗 19 2-12細胞遷移試驗 19 三、結果 20 3-1 探討腸上皮化生病患的miRNA表現差異 20 3-2 miR-10b-5p、miR-200b-3p、miR-200c-5p及miR-425-5p在胃癌細胞中高度表達 20 3-3 PMA處理AGS細胞後miR-425-5p的表達增加 21 3-4 PMA處理AGS細胞後miR-425-5p目標基因表達下調,另外亦在腸上皮化生的臨床組織中也有表現下調的現象 22 3-5 在AGS細胞中過表達miR-425-5p會抑制BANP及RABAC1的表達 23 3-6 BANP及RABAC1為miR-425-5p的直接目標基因 24 3-7 miR-425-5p會增加AGS胃癌細胞的生長速率 26 3-8 miR-425-5p會增加AGS胃癌細胞的遷移 27 四、討論 29 4-1 miRNA在腸上皮化生中的角色 29 4-2 miRNAs的目標基因在腸上皮化生中的影響 30 4-3 腸上皮化生分子機制的研究方法 31 4-4 從腸上皮化生找尋預測胃癌的生物標記 32 4-5 結論 33 參考文獻 35 圖表 47 附圖 71

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